Fluid vaccines and active principle vehicles containing a metabolizable oil

ABSTRACT

The present invention relates to an injectable emulsion, usable as a vaccine or active principle vehicle, which has an oily continuous phase with a good tolerance under normal conditions of injection into a human or an animal, said emulsion being characterized by a viscosity of less than 800 mPas at 20° C. and consisting of at least 
     20 to 90% of an oily adjuvant which is liquid at 4° C. and contains at least 
     one or more appropriate and pharmaceutically acceptable emulsifiers and 
     a mixture, in any proportions, of one or more metabolizable oils with one or more non-metabolizable oils, and 
     10 to 80% of a hydrophilic phase containing the active principles or antigens.

The present invention relates to novel compositions of fluid vaccinesand injectable substances, containing a totally or partiallymetabolizable oily phase, which can be used in veterinary or humanmedicine.

Vaccination is a means of combating infections by prevention, the aim ofwhich is to alert the immune defense system by the injection ofantigens, which are often very specific fractions of the viral orbacterial walls.

Advances in genetic engineering are such that the antigens available aremore and more pure or even totally synthetic. Although this purity is anundeniable asset from the point of view of the safety of the vaccine, itis generally accompanied by a decrease in the immunological efficacy.

There is therefore an ever-increasing demand for immunity adjuvantsmaking it possible to enhance the responses of the immune system to anantigen. However, the presence of these adjuvants must not compromisethe high safety of modern antigen preparations.

A very large number of immunity adjuvants have been described, but onlya few of them are used at the industrial vaccine stage and only aluminumhydroxide and phosphate are authorized in human medicine.

It is nevertheless acknowledged that "oily" vaccines, in which theantigen medium is emulsified with a mineral oil containing anemulsifier, are the most effective, especially when these vaccines areof the W/O type.

The adjuvant resulting from the association of mineral oil with amannitol ester is known as Freund's incomplete adjuvant (FIA) in theliterature; it has the following composition:

    ______________________________________                                        fluid mineral oil (Bayol F type):                                                                   85%                                                     mannide monooleate (Arlacel A):                                                                     15%                                                     ______________________________________                                    

The difference between FIA and FCA, Freund's "complete" adjuvant, isthat the latter contains, in addition to the above-mentioned components,a tuberculin mycobacterium which potentiates the immune effect.

These adjuvants FIA and FCA, which have been known for a long time, arestill the reference products for laboratory immunology studiesthroughout the world.

In particular, they are found associated with novel injectableadjuvants, such as the muramyl dipeptides (MDP), or antigens obtained bychemical or genetic synthesis (VP1, etc.).

The industrial use of vaccines containing FIA and FCA is limited,however, because of the difficulty of using them in injectable vaccinepreparations and their poor tolerance by the subjects vaccinatedtherewith.

In fact, it is known that oily emulsions obtained from FIA and FCA areviscous, like mayonnaise, and release oil. Consequently, thesepreparations are difficult to inject by means of syringes with a needleof small diameter (0.2 mm).

Furthermore, at the site of injection into animals, they create localreactions with edemas and abscesses, which are not acceptable to theHealth Authorities, making the animal unsuitable for human consumption.

Diphtheria vaccine preparations made with FCA have even caused seriousintolerances and abortion syndromes when injected into women, which haveprohibited the general use of these adjuvants in humans.

An Example of a foot-and-mouth disease vaccine containing FIA isdescribed below in order to demonstrate the immunological efficacy, thephysicochemical properties and the poor tolerance of this type ofpreparation.

EXAMPLE 1

A vaccine preparation for the treatment of foot-and-mouth disease incattle is made from Freund's incomplete adjuvant (FIA) by mixing onepart of FIA and one part of inactivated antigen medium, with mechanicalagitation.

The antigen medium was prepared with particular care during the culture,purification and concentration of the antigens, which are diluted in anEagle-type medium in the presence of phosphate buffer.

The preparation therefore has the following composition (weight/weight):

    ______________________________________                                        Antigen medium          50%                                                   Mineral oil             42.5%                                                 Mannide monooleate       7.5%                                                 ______________________________________                                    

The physicochemical characteristics obtained for this preparation are asfollows:

    ______________________________________                                        Conductivity at 20° C.:                                                                   12 microsiemens                                            Viscosity at 20° C.:                                                                      6500 centistokes                                           pH:                7.6                                                        Stability:         release of oil on                                                             storage at 4° C.                                    Microscopic appearance:                                                                          droplets larger than 2                                                        μm                                                      ______________________________________                                    

The immunological efficacy tests on cattle show a good immune responsewith persistent production of circulating antibodies 30 days afterintramuscular vaccination.

Experimental Section

Results of immunity of cattle to foot-and-mouth disease

oily vaccines with FIA - dose injected 5 ml

the results (EPP) are expressed as the percentage of the number ofanimals protected against viruses of valencies O, A and C, for a periodof 3 months, with vaccines from several batches, stored at 4° C.

                  TABLE 1                                                         ______________________________________                                                    Vaccine n° 1 stored                                                                  Vaccine n° 2 stored                          EPP*        for 1 month at 4° C.                                                                 for 24 months at 4° C.                       ______________________________________                                        Virus O                                                                              30 days* 79            77                                                     90 days  60            76                                              A      30 days  85            80                                                     90 days  70            69                                              C      30 days  93            95                                                     90 days  72            88                                              ______________________________________                                         *measurement of the protection 30 and 90 days after vaccination for           determining the prolonged effect of the immunity                         

Microscopic examination of the toxic effects on animals of the vaccinepreparation of the point of injection shows the substantial presence ofnodules and granulomas and even open abscesses. The extent of theinjured regions is considerable.

Histological examination shows the presence of oily residues in thetissues and an appreciable increase in the size of the external ganglia.

To improve the injectability of such oily vaccines, a known techniqueconsists of incorporating a small proportion of a hydrophilicemulsifier, namely polysorbate 80 (described in the pharmacopeias), inthe antigen medium.

The presence of this product substantially reduces the viscosity ofvaccines, as shown in Example 2.

However, it is known that polysorbate 80, which is used in biochemistryas a lipid dispersant, attacks cell walls and hence is potentiallytoxic.

EXAMPLE 2

The vaccine preparation made in Example 1 was modified by adding 1% ofpolysorbate 80 to the FIA.

The composition of the preparation becomes:

    ______________________________________                                        Antigen medium:         49.5%                                                 Mineral oil:            42.5%                                                 Mannide monooleate:      7.5%                                                 Polysorbate 80:          0.5%                                                 ______________________________________                                    

The physicochemical characteristics obtained are as follows:

    ______________________________________                                        Conductivity at 20° C.:                                                                    10 microsiemens                                           Viscosity at 20° C.:                                                                       350 centistokes                                           pH:                 7.6                                                       Stability:          slight ring of oil                                                            on storage at 4° C.                                ______________________________________                                    

The measurement of the immune response to intramuscular injection incattle is essentially equivalent to that obtained with the preparationof Example 1 when using a preparation according to Example 2 which wasmade less than 1 month earlier.

By contrast, when using a preparation according to Example 2 which wasmade 1 year earlier and has been stored at 4° C. under sterileconditions, it is found that there has been a deterioration in theantigen phase and a decrease in the immune response with a lower titerof circulating antibodies.

These results are explained by the presence of the hydrophilicemulsifier polysorbate 80 (characterized by an HLB of 15), which has awetting effect on the cellular envelopes of the antigens and solubilizesthe surface proteins, the result of which is to denature them and modifytheir ability to create antibodies.

The mechanism of this effect of hydrophilic surfactants onmicroorganisms is known and is described in particular in theliterature.

As regards the results of toxicity to animals by injection, thephenomena observed are similar to those obtained with the preparation ofExample 1 and are characteristic of poor tolerance of the vaccine.

Vaccine preparations made according to the method of Example 2, in whichFIA is associated with a hydrophilic emulsifier, are commerciallyavailable. Belgian patent A-648053 in the name of Philips describes abrucellosis vaccine which is made under similar conditions and which hasan appreciably reduced viscosity compared with the preparations madeusing FIA alone.

The use of metabolizable oils, such as vegetable oils, squalene andsqualane, in place of mineral oils has made it possible in certain casesto obtain vaccines of the W/O type which are tolerated much better.Weilbel et al. only observe small nodules of 3 to 4 mm in humansvaccinated with an influenza vaccine containing peanut oil (Pro. Soc.Exp. Biol. Med. 43, 1053-1056 (1973)), although the immune responsesrecorded are generally weaker and the protection shorter with this typeof vaccine than with vaccines containing mineral oil. This can beexplained by a more rapid degradation of the vaccine in the organism.

The Examples reported in British patent 1 081 796 (1965) in the name ofL. B. HOLT are very representative of this phenomenon (Table A).

                                      TABLE A                                     __________________________________________________________________________    Change in the antibody titer with 2 oily vaccines                             containing FIA or sweet-almond oil as adjuvant                                        ANTIBODY TITER                                                                DIPHTHERIA                                                                              TETANUS                                                             TOXOID    TOXOID     B. PERTUSSIS                                     ANTIGEN 6 weeks                                                                            13 weeks                                                                           6 weeks                                                                             13 weeks                                                                           6 weeks                                                                            13 weeks                                    __________________________________________________________________________    ADJUVANT                                                                              0.32 0.26 5.0   0.41 20   431                                         FIA                                                                           vegetable oil +                                                                       0.10 0.07  0.30 0.20 20    31                                         mannide                                                                       monooleate                                                                    __________________________________________________________________________

Another particularly disadvantageous characteristic of these vaccinescontaining metabolizable oil is their viscosity, which makes themdifficult and painful to inject. The vaccines, containing squalene orsqualane as adjuvant, which are described in European patent 0 117 934have a viscosity greater than 4000 mPas.

Likewise, those described in the article by WOODHOUR (Pro. Soc. Exp.Biol. Med. 116, 516-523, 1964), containing peanut oil and aluminummonostearate, are presented as highly viscous water-in-oil emulsions.

It has been found, quite unexpectedly, that the addition of ametabolizable oil to vaccines containing a mineral oil (or, in general,a non-metabolizable oil) makes it possible considerably to improve theirtolerance without significantly modifying their immunological efficacy,provided that the vaccines obtained are stable and very fluid.

The use of weakly hydrophilic, non-toxic emulsifiers, obtained fromliquid fatty acids, mannitol or glycerol, has made it possible to obtainthese totally or partially metabolizable, stable, fluid preparations.

The vaccines (or injectable preparations) of the invention arecharacterized by 4 important physicochemical parameters:

fluidity: the viscosity, measured by a BROOKFIELD-type viscometer, mustbe less than 800 centistokes at 20° C. for the vaccine to be injectablethrough a syringe needle with a diameter of 0.2 mm;

stability: the emulsified preparation must not undergo phase separation,i.e. must not release either the internal antigen phase or the oilyphase under storage conditions which are normal for this type ofproduct;

conductivity: this determines the oily or aqueous character of thecontinuous phase of the emulsion; for values below about 20microsiemens, an oily continuous phase is obtained at room temperature;

microscopic appearance: the sizes of dispersed droplets of antigen phaseare less than 10 μm and distributed over a narrow range.

Apart from the antigen medium or the active principles specific to eachinjectable preparation, the other constituents (oils, emulsifiers) mustsatisfy precise criteria defined below.

Definition of the Oil

The fluid oils are selected from mineral, vegetable or animal oils knownfor their low toxicity. They must be liquid at the storage temperature(+4° C.) or they must at least give liquid emulsions at thistemperature. Mineral oils which have a linear chain with more than 16carbon atoms and are devoid of aromatic compounds will be chosen inparticular.

Known examples are MARCOL 52 (produced by ESSO France) and DRAKEOL 6VR(produced by PENRECO USA).

It is also possible to use synthetic mineral oils such as polyisobutenesor polyisoprenes.

Among the vegetable oils, unsaturated oils rich in oleic acid, which arebiodegradable and known for their immunogenic power, will be chosen,examples being peanut oil, olive oil, sesame oil, soy bean oil,wheatgerm oil, etc.

As regards the animal oils, the same criteria of tolerance andimmunological efficacy make it possible to use, for example, squalene,squalane and spermaceti oil.

It is advantageous, within the framework of the present invention, touse a mixture of the aforementioned oils.

Definition of the Emulsifier System

The emulsifier system must be adapted to give fluid and stableinjectable preparations of the W/O type.

It is composed of one or more surfactants which, when mixed together,have a lipophilic or weakly hydrophilic character with an HLB(hydrophile-lipophile balance) of between 2 and 9.

The emulsifiers are preferably obtained by condensing a fatty acid whichis liquid at 20° C. with a sugar (mannitol, glucose, sucrose) orglycerol. The preferred fatty acids are those having at least 16 carbonatoms, in particular oleic, linoleic, ricinoleic and cetostearic acids.

It is preferred to use mannitol esters, especially the oleates, obtainedunder particular conditions of synthesis by dehydrating thepolyhydroxylated hydrocarbon chain of mannitol, which cyclizes in the1-4 or 2-6 positions. The hydrophilicity of the esters obtained can bemodified by the grafting of hydrophilic groups such as an alcohol, apolyol, ethylene oxide, propylene oxide, a carboxylic acid, an amine, anamide, etc.

All the emulsifiers used must be pharmaceutically acceptable for use ininjectable formulations; in particular, they must be devoid of heavymetals and must have very low acid or peroxide numbers. It is alsodesirable that they should satisfy the standards of safety tests suchas, for example, the one described by S. S. BERLIN (Annals of Allergy20, 473, 1962).

It is also desirable that the emulsifiers used should form a homogenous,clear and stable phase with the chosen oil, which phase will beemulsified with the aqueous medium appropriate to each injectablepreparation.

The emulsifiers described in Table B meet the above demands. They areused in the injectable preparations described in Examples 3 to 9 to formfluid and stable emulsions.

                                      TABLE B                                     __________________________________________________________________________    Physicochemical characteristics of emulsifiers which can be used in the       invention                                                                     Emulsifier      Reference  1       2       3       4    5                     __________________________________________________________________________    Solubility*                                                                         Oil       S          S       S       S       S    S                           Water     I          D       I       I       I    D                     HLB (approximate)                                                                             2.6        5       6.5     3       2    3                     Refractive index                                                                              1.4750-1.4765                                                                            1.4748-1.4758                                                                         1.4740-1.4750                                                                         --      --   --                    Hydroxyl number  89-100     90-105  90-110  87-100 20-90                                                                              --                    Saponification number                                                                         164-172    147-160 120-140 157-170 166-186                                                                            --                    Purity                                                                        Heavy metals ppm                                                                              20 max.    20 max. 20 max. 20 max. 20 max.                                                                            20 max.               Peroxides mmol/kg                                                                              2 max.     2 max.  2 max.  3 max.  3 max.                                                                             3 max.               Acid number      1 max.     1 max.  1 max.  1 max.  3 max.                                                                             2 max.               Water %          2 max.     2 max.  2 max.  2 max.  2 max.                                                                             2 max.               Chemical definition or tradename                                                              Mannide monooleate                                                                       Montanide 888                                                                         Montanide 103                                                                         Montanide 9208                                                                        Mannide                                                                            Purified soya                         Montanide 80 1                                                                           1       1       1       dioleate                                                                           lecithin                                                                      LIPOID S              __________________________________________________________________________                                                            75                     S = Soluble  I = Insoluble  D = Dispersible                                   (1) registered trademark of SEPPIC France                                     (2) registered trademark of LIPOID FRG                                   

EXAMPLE 3

This Example shows the importance of the fluidity of the vaccines ontheir tolerance by animals.

Two AUJESZKY's disease vaccines for pigs are prepared which contain thesame antigen medium inactivated with β-propiolactone. Thecharacteristics of vaccine A according to the invention and of vaccine B(containing an adjuvant of the FIA type) are mentioned in Table 3-1. Asis apparent from Table 3-1, and as previously described, the range ofviscosities is about 37 mPas to 800 mPas.

Vaccine A is characterized by a low viscosity and a good injectability:it contains emulsifier 2 described in Table B.

Both vaccines contain the same fluid mineral oil.

The experiment was performed on 2 groups of 6 pigs to which 2 times 2 mlof vaccine were administered intramuscularly at an interval of 15 days.Serological analysis and histological examination were carried out 59days after the first vaccination.

The results are collated in Table 8-2. It can be seen from this Tablethat the antibody titers are similar for the 2 vaccines, but thatvaccine A is tolerated much better than vaccine B, for which 2 pigsdisplayed abnormal behavior after each vaccination. Necroses andsuppurations at the sites of injection--which are totally unacceptableto the Health Authorities--are practically absent from group A. Themacrophagic granulomas and the inflammatory infiltrates observed areevidence of an intense immune reaction.

                  TABLE 3 - 1                                                     ______________________________________                                        Characteristics of Aujeszky's disease vaccines for pigs                                      Vaccine A Vaccine B                                            ______________________________________                                        Characteristics of the adjuvant                                               Composition      mineral oil:                                                                              mineral oil:                                                      90%         89%                                                               emulsifier  mannide mono-                                                     2: 10%      oleate: 11%                                      Viscosity (mPa · s)                                                                   35          40                                               Refractive index 1-459       1-461                                            Characteristics of the vaccine                                                Composition % w/w                                                                              adjuvant: 70                                                                              adjuvant: 50                                                      medium: 30  medium: 50                                       Viscosity (BROOKFIELD                                                                          37 mPa · s                                                                       1870 mPa · s                            M2V12)                                                                        Emulsion type    W/O         W/O                                              Conductivity     0.2 μS   0.4 μS                                        Stability at 4° C.                                                                      >12 months  >12 months                                       Microscopic appearance                                                                         1 μ drops                                                                              1-5 μ drops                                   ______________________________________                                    

                  TABLE 3 - 2                                                     ______________________________________                                        Vaccination results                                                                            Vaccine A                                                                             Vaccine B                                            ______________________________________                                        Serology                                                                      Antibody titer (log base 2)                                                                        2.5       2.0                                            (mean over 5 animals)                                                         Histology                                                                     (frequency over 6 pigs)                                                       Necrosis           1         3                                                Suppuration        0         3                                                Fibrosis           5         5                                                Muscular atrophy   5         6                                                Macrophagic or lymphocytic                                                                       5         4                                                granulomas                                                                    Diffuse inflammatory infiltrate                                                                  2         2                                                General reaction                                                              (modification of behavior)                                                                       0         2                                                ______________________________________                                    

EXAMPLE 4

A foot-and-mouth disease vaccine for cattle is prepared by mixing onepart of adjuvant containing emulsifier 1 and one part of the inactivatedantigen medium, with mechanical agitation. The adjuvant contains 5% of avegetable oil.

The physicochemical characteristics of the adjuvant and of the vaccineobtained are given below (Table 4-1).

The immunological efficacy test gives the results summarized in Table4-2, which can be compared with the preparation of Example 1.

The activity is maintained in preparations stored for up to 2 years.

The tolerance of the preparation injected intramuscularly into cattle isgood. Histological examination reveals a slight inflammation of thetissues at the point of injection, with the presence of only a fewnodules and granulomas. Microscopic absorption of the inflammation andmicroscopic disappearance of the injected product are observed within 15days of the injection.

                  TABLE 4 - 1                                                     ______________________________________                                        Characteristics of foot-and-mouth disease vaccine                             ______________________________________                                        Characteristics of the adjuvant                                               Composition      peanut-oil     5%                                                             fluid mineral oil                                                                           84%                                                             emulsifier 1  11%                                            Appearance    stable, light yellow, clear oily liquid                         Viscosity     40 mPa · s                                             Acid number   0.1                                                             Refractive index                                                                            about 1.460                                                     Density       0.85                                                            Characteristics of the foot-and-mouth disease vaccine                         Conductivity at 20° C.                                                               1.5 microsiemens                                                Viscosity at 20° C.                                                                  250 centistokes                                                 Stability at 4° C.                                                                   very slight ring of oil                                         Microscopic appearance                                                                      droplets smaller than 1                                         Centrifugation                                                                              no separation after 30 min at 3000 rpm                          ______________________________________                                    

                  TABLE 4 - 2                                                     ______________________________________                                         Results of immunity of cattle to foot-and-mouth disease                      Dose injected: 5 ml                                                           The results (EPP) are expressed as the percentage of animals                  protected against viruses of valencies O, A and C for a period                of 3 months following vaccination with vaccines from several                  batches, stored for several months at 4° C..                                    Vaccine   Vaccine   Vaccine Vaccine                                           batch n° 3                                                                       batch n° 3                                                                       batch n° 4                                                                     batch n° 4                                 stored for                                                                              stored for                                                                              stored for                                                                            stored for                               EPP      1 month   24 months 1 month 3 months                                 ______________________________________                                        Virus 30 d   90        99      99      90                                     O     90 d   88        99      99      86                                     Virus 30 d   86        92      97      82                                     A     90 d   93        90      99      85                                     Virus 30 d   97        99      99      97                                     C     90 d   92        97      99      85                                     ______________________________________                                    

EXAMPLE 5

Mice are vaccinated with vaccines consisting of

an antigen medium containing 100 μg/ml of bovine albumin, and

an oily adjuvant containing either a synthetic oil (vaccine 5A),

or a synthetic oil and a vegetable oil (vaccine 5B).

These two vaccines are fluid and stable. Their physicochemicalcharacteristics are given in Table 5-1.

Groups of 10 SWISS mice are vaccinated with 0.1 ml of these vaccines. Acontrol group receives the antigen medium without adjuvant.

The antibody titers, measured over time by an ELISA technique, are givenin Table 5-2. It is found that the two vaccines with adjuvant behave inthe same way even though vaccine 5B contains a metabolizable oil, andare distinctly more effective than the vaccine without adjuvant. Therapid appearance of the antibodies with the vaccine containing themetabolizable oil will be noted.

                  TABLE 5 - 1                                                     ______________________________________                                        Physicochemical characteristics of BSA vaccine                                               Vaccine A Vaccine B                                            ______________________________________                                        Characteristics of the adjuvant                                               Composition      polyisobutene:                                                                            polyisobutene:                                                    89%         44%                                                               emulsifier  sweet-almond                                                      2: 11%      oil: 45%                                                                      emulsifier                                                                    3: 11%                                           Viscosity (mPa · s)                                                                   45          50                                               Appearance       clear colorless                                                                           clear yellow                                                      liquid      liquid                                           Characteristics of the vaccine                                                Emulsion type    W/O         W/O                                              Conductivity at 20° C.                                                                  0.9 μS   1.2 μS                                        Viscosity mPa · s at 20° C.                                                    100         130                                              Stability        >12 months  >12 months                                       Appearance       homogeneous homogeneous                                                       emulsion    emulsion                                         ______________________________________                                    

                  TABLE 5 - 2                                                     ______________________________________                                        Antibody titer in mice vaccinated with BSA vaccines                           Days after vaccination                                                                         14     28       56   125                                     ______________________________________                                        Vaccine without adjuvant                                                                        60     157      102  105                                    Vaccine 5A        590   2420     3800 4560                                    Vaccine 5B       1400   2540     3760 4640                                    ______________________________________                                    

EXAMPLE 6

An injectable preparation containing hormonal active principles is madeby mixing one part of an aqueous suspension and one part of an oilyadjuvant containing metabolizable oils (squalene and sweet-almond oil:84%), a non-metabolizable oil (squalane: 5%) and emulsifier 3 (11%).

The vaccine obtained has the following characteristics:

    ______________________________________                                        EMULSION TYPE           W/O                                                   CONDUCTIVITY            0.14 μS                                            VISCOSITY               305 mPa · s                                  STABILITY at 4° C. and 20° C.                                                           >6 months                                             ______________________________________                                    

These characteristics, in particular the viscosity and stability, canonly be obtained by virtue of the specific formulation of the oilyadjuvant. In fact, a vaccine which has the same water/oil ratio but isformulated on the basis of squalene and traditional mannitol monooleateis very viscous (4400 mPas), extremely difficult and painful to injectand of low stability (substantial exudation of oil at 4° and 20° C.).

The preparation according to the invention has a sufficient efficacyand, in primates, causes only slight redness at the point of injection,which disappears after a few days.

EXAMPLE 7

An Aujeszky's disease vaccine is prepared by mixing 30 parts of aninactivated vital medium with a titer of 2.10⁹ DCP 50/ml and 70 parts ofan oily adjuvant containing a vegetable oil and emulsifier 3.

Table 7-1 gives the characteristics of the adjuvant and of the vaccineobtained. 2 ml of vaccine are injected into a group of 5 pigs and thisis followed by a booster after 15 days. A resistance test is performed15 days after the booster. Slaughter and histological examination tookplace 5 weeks after the booster.

The results of the experiment show a very good tolerance of the vaccinein all areas. These results can be compared with those obtained for aconventional oily vaccine containing an adjuvant of the FIA type andpresented in Example 3 (vaccine B).

It should be noted in particular that there is an absence of excessivehyperthermia, necroses and suppurations and a presence of discretemacrophagic granulomas, representing the immune response and confirmingthe good resistance to the infection test.

                  TABLE 7 - 1                                                     ______________________________________                                        Physicochemical characteristics of the vaccine and its adjuvant               ______________________________________                                        Characteristics of the adjuvant                                               Composition      fluid mineral oil                                                                            44%                                                            refined peanut oil                                                                           44%                                                            emulsifier 1   12%                                           Appearance       homogeneous, stable, clear,                                                   straw yellow oily liquid                                     Viscosity at 20° C.                                                                     20 mPa · s                                          Refractive index 1.466                                                        Hydroxyl number  13                                                           Saponification number                                                                          105                                                          Characteristics of the vaccine                                                Emulsion type    W/O                                                          Conductivity at 20° C.                                                                  1.9 μS                                                    Viscosity at 20° C.                                                                     38 mPa · s                                          Microscopic appearance                                                                         homogeneous - 1 μm drops                                  Stability at 4° C.                                                                      >12 months                                                   ______________________________________                                    

                  TABLE 7 - 2                                                     ______________________________________                                        Experimental results of pig vaccination                                       ______________________________________                                        Hyperthermia                                                                             at the vaccination 0                                                          at the booster     brief in 2 animals                                         at the test        intense but brief                                                             (<48 h)                                         General reactions                                                                        at the vaccination slight inflammation                                        at the booster     at the point of                                                               injection, disappearing                                                       in 48 h                                                    at the test        0                                               Mortality (after the test)                                                                        0                                                         Histology (frequency over 5 pigs)                                             Necrosis            0                                                         Suppuration         0                                                         Fibrosis            0                                                         Muscular atrophy    5 (slight)                                                Macrophagic granulomas                                                                            5 (disseminated, discrete)                                ______________________________________                                    

EXAMPLE 8

The same experiment as that described in Example 7 was carried out witha vaccine containing 70 parts of an adjuvant composed of squalane, afatty acid ester and a slightly hydrophilic emulsifier described underreference 2 in Table B, and 80 parts of Aujeszky's antigen medium. Thevaccine obtained is very fluid and is easy to inject. Its performancedata and characteristics are given in Tables 8-1 and 8-2.

It will again be noted that there is an absence of substantialintolerance reactions compared with the conventional vaccine of Example1B.

                  TABLE 8 - 1                                                     ______________________________________                                        Physicochemical characteristics of Aujeszky's disease vaccine                 for pigs                                                                      ______________________________________                                        Characteristics of the adjuvant                                               Composition     ethyl oleate   6%                                                             squalane      83%                                                             emulsifier 2  11%                                             Appearance      very light yellow, transparent                                                clear liquid                                                  Viscosity at 20° C.                                                                    20 mPa · s                                           Refractive index                                                                              about 1.492                                                   Characteristics of the vaccine                                                Emulsion type   W/O                                                           Viscosity at 20° C.                                                                    88 mPa · s                                           Conductivity at 20° C.                                                                 0.03 μS                                                    Stability at 4° C.                                                                     slight exudation of oil                                       ______________________________________                                    

                  TABLE 8 - 2                                                     ______________________________________                                        Experimental results of pig vaccination                                       ______________________________________                                        Hyperthermia                                                                              at the vaccination                                                                          very slight                                                     at the booster                                                                              >41° C., 24 h                                            at the test   intense but brief                                   General reactions                                                                         at the vaccination                                                                          0                                                               at the booster                                                                              slight inflammation                                                           at the site of                                                                injection                                                       at the test   not pronounced                                      Mortality (after the test)                                                                            0                                                     Histology (frequency over 5 pigs)                                             Caseous abscesses       2                                                     Suppuration             0                                                     Fibrosis                1                                                     Muscular atrophy        5                                                     Macrophagic granulomas  3                                                     ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Results of colibacillus vaccination in rabbits                                ______________________________________                                                  days                                                                Serology*   0      15     24  booster                                                                              42    84                                 ______________________________________                                        Vaccine of  0      9.5    11  ↓                                                                             11.3  10.6                               Example 9                                                                     Vaccine with FIA                                                                          0      6       7         8     8                                  ______________________________________                                        Reactions   days                                                              observed**  1      4      5    19    ↓                                                                          31    50                             ______________________________________                                        Vaccine of  0      20     27    0        35    0                              Example 9                                                                     Vaccine with FIA                                                                          0      30     40   10        60   10                              ______________________________________                                         *Antibody titer expressed as log base 2 of the last agglutinating dilutio      geometric mean                                                               **Mean diameters of the erythemas observed (mm) at the point of injection

EXAMPLE 9

A colibacillus vaccine was prepared for vaccination in rabbits.

The antigen medium is a suspension of attachment factor K 88 ab inisotonic solution at a concentration such that the OD of this suspensionis 0.5 at 540 nm.

The adjuvant used is the one described in Example 7 and in Table 7-1.

The vaccine contains 1/3 of antigen medium and 2/3 of adjuvant. It hasthe following characteristics:

    ______________________________________                                        EMULSION TYPE          W/O                                                    CONDUCTIVITY at 20° C.                                                                        2.0 μS                                              VISCOSITY at 20° C.                                                                           40 mPa · s                                    ______________________________________                                    

6-week-old New Zealand rabbits are vaccinated with 1 ml of vaccine and abooster is then given after 29 days.

The antibody titers are measured by a micro-agglutination technique.

Table 9 gives the results obtained with this vaccine and with aconventional oily vaccine formulated with Freund's incomplete adjuvant(50/50 formula; viscosity>3000 mPas).

The antibody titers with the vaccine according to the invention aregreater than or equal to those measured with the conventional vaccine.The reactions at the site of injection are very substantial with theconventional vaccine, especially after the booster.

They are much weaker and regress more rapidly with the vaccine of thepresent Example.

EXAMPLE 10

A Newcastle disease vaccine preparation for fowls was made using anadjuvant consisting of

    ______________________________________                                        DRAKEOL 6VR fluid mineral oil                                                                        82%                                                    Soy bean oil            5%                                                    Emulsifier 2           13%                                                    ______________________________________                                    

This clear liquid adjuvant has the following characteristics:

Acid number below 0.5

Hydroxyl number of about 11

Refractive index of 1,459

The vaccine is prepared by mixing 7 parts of adjuvant and 3 parts ofallantoid medium containing inactivated Newcastle antigens, withmechanical agitation.

The vaccine has the following physicochemical characteristics:

    ______________________________________                                        Conductivity at 20° C.                                                                   below 1 microsiemens                                        Viscosity at 20° C.                                                                      45 centistokes                                              Stability         no release of oil or                                                          phase separation of the                                                       medium at 4° C. after 3                                                months                                                      ______________________________________                                    

The preparation also remains stable at 37° C. for more than a week.

Vaccines stored for 12 months at 4° C. and for 1 month at roomtemperature, as well as a control consisting of the inactivated viralantigens without adjuvant and a live virus vaccine, are injected intothe animals.

The study is carried out on 3 series of ten 4-week-old EOPS chickens ofthe LEGHORN type. The injection is given subcutaneously in the neckregion with a dose of 0.5 ml.

The immunological efficacy is measured by titrating the hemagglutinationinhibition.

Blood samples are taken from the wing vein of the vaccinated fowl attimes Two, 4, 8, 12 and 18 weeks and the hemagglutinin content ismeasured.

    ______________________________________                                        Titer of hemagglutination inhibition                                                      Two  4 wks   8 wks   12 wks                                                                              18 wks                                 ______________________________________                                        1 (control)    10     9       10    8     8                                   2 (vaccine according                                                                        210    230     190   150   160                                  to the invention)                                                             3 (live virus 305    210     120    70    50                                  vaccine)                                                                      ______________________________________                                    

It is seen that the immunological activity obtained with the vaccine ofthe invention is superior to that of the control.

Contrary to what is observed with the live virus vaccine, the activitypersists after more than 4 months, which makes it possible to ensureimmune protection of chickens against Newcastle disease throughout theirlife.

From the point of view of toxicity, no mortality of the fowls vaccinatedwith the vaccine according to the invention is found, whereas a numberof chickens (5%) die shortly after injection with the live virusvaccine.

We claim:
 1. A parenteral vaccine in the form of an emulsion whichcomprises on a weight basis:10 to 80% of a hydrophilic phase containingone or more antigens; 20 to 90% of an oil adjuvant in the form of ahomogenous and stable phase which comprises:one or more metabolizableoils selected from oils of vegetable origin, oils of animal origin,synthetic oil in which the mean number of carbon atoms is at least 16;one or more non-metabolizable mineral oils representing from 2 to 95% ofthe oil adjuvant and selected from the group consisting of mineral oilshaving a mean number of carbon atoms equal to at least 16; one or morenon-toxic, pharmaceutically acceptable emulsifiers which, when mixedtogether have a lipophilic or weakly hydrophilic character with an HLB(hydrophile-lipophile balance) of between 2 and 9, said emulsifiersbeing selected from the group consisting of:mannitol esters; mannitolesters grafted with hydrophilic groups selected from the groupconsisting of alcohol, polyol, ethylene oxide, propylene oxide,carboxylic acid, amine and amide; said preparation being stable andhaving a viscosity of between about 37 mPas to 800 mPas at 20° C.wherein said emulsion provides improved tolerance of said parentalvaccine without altering the immunological efficacy of said parenteralvaccine.
 2. A preparation according to claim 1, in which themetabolizable oil is of vegetable origin.
 3. A preparation according toclaim 1, in which the metabolizable oil is of animal origin.
 4. Apreparation according to claim 1 in which the metabolizable oil is anester of a fatty acid containing from 12 to 24 carbon atoms and analcohol, or an ester of a fatty alcohol containing from12 to 24 carbonatoms and an acid.
 5. A preparation according to claim 1, wherein saidmannitol esters are mannitol oleates.
 6. A parenteral vaccinepreparation in the form of an emulsion which comprises on a weightbasis:10 to 80% of a hydrophilic phase containing one or more antigens;20 to 90% of an oily adjuvant in the form of a homogenous and stablephase which comprises:one or more metabolizable oils selected from thegroup consisting of oils of vegetable origin, oils of animal origin,synthetic oil in which the mean number of carbon atoms is at least 16;one or more non-metabolizable mineral oils representing from 2 to 95 %of the oily adjuvant and selected from mineral oils having a mean numberof carbon atoms equal to at least 16; and one or more non-toxic,pharmaceutically acceptable emulsifiers which, when mixed together, havea lipophilic or weakly hydrophilic character with an HLB(hydrophile-lipophile balance) of between 2 and 9, said emulsifiersbeing selected from the group consisting of:esters of fatty acids andsugar; esters of fatty acids and sugar grafted with hydrophilic groupsselected from the group consisting of alcohol, polyol, ethylene oxide,propylene oxide, carboxylic acid, amine and amide; esters of fatty acidsand glycerol; and esters of fatty acids and glycerol grafted withhydrophilic groups selected from the group consisting of alcohol,polyol, ethylene oxide, propylene oxide, carboxylic acid, amine andamide; said preparation being stable and having a viscosity of betweenabout 37 mPas and 800 mPas at 20° C. wherein said emulsion providesimproved tolerance of said parental vaccine without altering theimmunological efficacy of said parenteral vaccine.
 7. A preparationaccording to claim 6, wherein said sugar is selected from the groupconsisting of mannitol, glucose and sucrose.
 8. A preparation accordingto claim 6, wherein said fatty acid is selected from the groupconsisting of oleic acid, linoleic acid, ricinoleic acid and cetostearicacid.
 9. A preparation according to claim 6, wherein said emulsifiersare selected from the group consisting of mannitol oleates, mannitololeates grafted with hydrophilic groups, mannitol ricinoleates, mannitolricinoleates grafted with hydrophilic groups, said hydrophilic groupsbeing selected from the groups consisting of alcohol, polyol, ethyleneoxide, propylene oxide, carboxylic acid, amine and amide.
 10. Aparenteral vaccine preparation in the form of an emulsion whichcomprises on a weight basis:10 to 80% of a hydrophilic phase containingone or more antigens; 20 to 90% of an oily adjuvant in the form of ahomogeneous and stable phase which comprises:one or more metabolizableoils selected from oils of vegetable origin, oils of animal origin,synthetic oil of the alkane, alkene or alkyne type in which the meannumber of carbon atoms is at least 16; one or more non-metabolizablemineral oils representing from 10 to 50% of the oily adjuvant andselected from the group consisting of mineral oils having a mean numberof carbon atoms equal to at least 16; and one or more non-toxic,pharmaceutically acceptable emulsifiers which, when mixed together, havea lipophilic or weakly hydrophilic character with an HLB(hydrophile-lipophile balance) of between 2 and 9, said emulsifiersbeing selected from the group consisting of:esters of fatty acids andsugar; esters of fatty acids and sugar grafted with hydrophilic groupsselected from the group consisting of alcohol, polyol, ethylene oxide,propylene oxide, carboxylic acid, amine and amide; esters of fatty acidsand glycerol; and esters of fatty acids and glycerol grafted withhydrophilic groups selected from the group, consisting of alcohol,polyol, ethylene oxide, propylene oxide, carboxylic acid, amine andamide; said preparation being stable and having a viscosity of betweenabout 37 mPas and 800 mPas at 20° C. wherein said emulsion providesimproved tolerance of said parental vaccine without altering theimmunological efficacy of said parenteral vaccine.
 11. A preparationaccording to claim 10, wherein said sugar is selected from the groupconsisting of mannitol, glucose and sucrose.
 12. A preparation accordingto claim 10, wherein said fatty acid is selected from the groupconsisting of oleic acid, linoleic acid, ricinoleic acid and cetostearicacid.
 13. A preparation according to claim 10, wherein said emulsifiersare selected from the group consisting of mannitol oleates, mannitololeates grafted with hydrophilic groups, mannitol ricinoleates, mannitolricinoleates grafted with hydrophilic groups, said hydrophilic groupsbeing selected from the groups consisting of alcohol, polyol, ethyleneoxide, propylene oxide, carboxylic acid, amine and amide.